Analyses of Porphyria Caused by Environmental Pollutants : Concerted Reaction and Tempo of Heme and Porphyrin Syntheses

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In eukaryotes, protein synthesis is terminated when cells face an emergency such as a shortage of amino acids, stress to the endoplasmic reticulum, and viral infection. Translation initiation in the red cell is regulated by eukaryotic initiation factor 2a (a-subunit of eukaryotic initiation factor 2 ; eIF2α) kinases or HRI (heme-regulated inhibitor). In the red cell, protein synthesis is terminated when cells face an emergency such as shortage of the heme iron complex. HRI senses and mediates responses to changes in the Fe(II)-protoporphyrin IX (heme) concentration. Under normal conditions when there is sufficient heme, HRI does not phosphorylate eIF2α, but when there is a shortage of heme, it acts to maintain the heme-to-globin ratio at 1:1 by phosphorylating eIF2a, which initiates the termination of protein synthesis. Although HRI is reported to sense the heme concentration in reticulocytes or red blood cells the molecular mechanisms, in particular the role of the N-terminal domain in its oligomerization, kinase function, and heme sensing, have remained elusive. In addition, little is known regarding how heme binds to the catalytic domain and regulates catalysis, the stoichiometry of heme binding, and which residues are axial ligands for the heme iron. In the present study, we attempted to elucidate how the concerted reactions of the heme and protein syntheses are coupled by generating the N-terminal truncated mutants, His mutants, Cys mutants and examining the catalytic function, heme affinities, and heme coordination structures. We also examined effects of environmentally polluted heavy metal cations on the kinase activities of HRI. It was found that NO enhances catalysis of HRI, NO recovers inhibited calysis by heme and bilirubin and heavy metal cations inhibit catalysis. The results obtained will be helpful to understand the molecular mechanism of porphyria caused by environmental pollutants.